Achieving hemostasis following vascular injury while avoiding excessive platelet accumulation requires that signaling is closely regulated in resting and activated platelets. Most platelet agonists work through G protein coupled receptors (GPCRs). Our goals are to dissect how GPCRs and G proteins can be regulated by GPCR kinases (GRKs) during platelet activation and thrombus formation, and to understand how dysfunctional regulation of GRKs may lead to thrombotic events and cardiovascular disease. In the past two decades, GRKs have been shown to play an important role in the heart by regulating GPCR signaling. Changes in GRK expression have been linked to many cardiovascular pathologies. However, the contribution of GRKs to platelet activation and the role of GRKs in hemostasis and thrombosis are unknown. Our hypothesis is that GRKs are critical negative regulators of platelet activation and thrombus formation. We base our hypothesis on preliminary studies from our lab showing that 1) The RGS-resistant G188S mutation in Gq prevents RGS protein binding but surprisingly increases GRK2 binding; 2) GRK2 binding is specific to Gq, but not to Gi2; 3) In contrast to enhanced Gi2 signaling in RGS-resistant Gi2(G184S) mutant platelets, there is decreased platelet activation in Gq(G188S) platelets; 4) Deletion of GRK2, GRK5 or GRK6 in platelets causes an increase in platelet activation. The hypothesis will be tested in three specific aims. In Aim 1, we will examine the role of G protein and GRKs interactions in regulating platelet function. In Aim 2, we will determine the role of GRK2 in hemostasis and thrombosis. we will be the first to characterize the functions of GRK2 in hemostasis and thrombosis and identify the non-canonical roles of GRK2 in platelets. In Aim 3, we will determine the role of GRK5/6 in hemostasis and thrombosis and explore the effect of two human GRK5 genetic variants on platelet function using CRISPR-Cas9 edited iPSCs (induced pluripotent stem cells). These proposed studies are innovative because we combine recent genome-wide association studies (GWAS) identified human GRK5 genetic variants, 4 newly generated mouse mutant lines and CRISPR-cas9 edited iPSC cells to study the uncharacterized role of GRKs in platelets. This study is significant because a critical gap in knowledge exists between unexplored functions of GRK family in platelets and their well-studied roles in the cardiovascular system. Through these studies, we will advance our understanding of the role of GRKs in cardiovascular health and disease, and the gained information may lead to new therapeutic options for the treatment of thrombotic and cardiovascular disorders.